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It is well appreciated that, differently from skeletal muscles, the heart contracts independently from neurogenic inputs. However, the speed and force of heartbeats are finely modulated during stresses, emotions, and daily activities, by the autonomic neurons (both parasympathetic and sympathetic) which highly innervate the myocardium. Despite this aspect of cardiac physiology has been known for long, research has only recently shed light on the biophysical mechanisms underlying the meticulous adaptation of heart activity to the needs of the organism. A conceptual advancement in this regard has come from the use of optogenetics, a revolutionary methodology which allows to control the activity of a given excitable cell type, with high specificity, temporal and spatial resolution, within intact tissues and organisms. The method, widely affirmed in the field of neuroscience, has more recently been exploited also in research on heart physiology and pathology, including the study of the mechanisms regulating heart rhythm. The last point is the object of this book chapter which, starting from the description of the physiology of heart rhythm automaticity and the neurogenic modulation of heart rate, makes an excursus on the theoretical basis of such biotechnology (with its advantages and limitations), and presents a series of examples in cardiac and neuro-cardiac optogenetics., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Key Points: The heart is innervated by a dense sympathetic neuron network which, in the short term, controls chronotropy and inotropy and, in the long term, regulates cardiomyocyte size. Acute neurogenic control of heart rate is achieved locally through direct neuro-cardiac coupling at specific junctional sites (neuro-cardiac junctions). The ventricular sympathetic network topology is well-defined and characteristic for each mammalian species. In the present study, we used cell size regulation to determine whether long-term modulation of cardiac structure is achieved via direct sympatho-cardiac coupling. Local density of cardiac innervation correlated with cell size throughout the myocardial walls in all mammalian species analysed, including humans. The data obtained suggest that constitutive neurogenic control of cardiomyocyte trophism occurs through direct intercellular signalling at neuro-cardiac junctions., Abstract: It is widely appreciated that sympathetic stimulation of the heart involves a sharp increase in beating rate and significant enhancement of contractility. We have previously shown that, in addition to these evident functions, sympathetic neurons (SNs) also provide trophic input to cardiomyocytes (CMs), regulating cell and organ size. More recently, we have demonstrated that cardiac neurons establish direct interactions with CMs, allowing neuro-cardiac communication to occur locally, with a 'quasi-synaptic' mechanism. Based on the evidence that cardiac SNs are unevenly distributed throughout the myocardial walls, we investigated the hypothesis that CM size distribution reflects the topology of neuronal density. In vitro analyses of SN/CM co-cultures, ex vivo confocal and multiphoton imaging in clarified hearts, and biochemical and molecular approaches were employed, in both rodent and human heart biopsies. In line with the trophic effect of SNs, and with local neuro-cardiac communication, CMs, directly contacted by SNs in co-cultures, were larger than the non-targeted ones. This property reflects the distribution of CM size throughout the ventricles of intact mouse heart, in which cells in the outer myocardial layers, which were contacted by more neuronal processes, were larger than those in the less innervated subendocardial region. Such differences disappeared upon genetic or pharmacological interference with the trophic SN/CM signalling axis. Remarkably, CM size followed the SN distribution pattern in other mammals, including humans. Our data suggest that both the acute and chronic influence of SNs on cardiac function and structure is enacted as a result of the establishment of specific intercellular neuro-cardiac junctions., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

The audience of basic and clinical scientists is familiar with the notion that the sympathetic nervous system controls heart function during stresses. However, evidence indicates that the neurogenic control of the heart spans from the maintenance of housekeeping functions in resting conditions to the recruitment of maximal performance, in the fight-or-flight responses, across a whole range of intermediate states. To perform such sophisticated functions, sympathetic ganglia integrate both peripheral and central inputs, and transmit information to the heart via 'motor' neurons, directly interacting with target cardiomyocytes. To date, the dynamics and mode of communication between these two cell types, which determine how neuronal information is adequately translated into the wide spectrum of cardiac responses, are still blurry. By combining the anatomical and structural information brought to light by recent imaging technologies and the functional evidence in cellular systems, we focus on the interface between neurons and cardiomyocytes, and advocate the existence of a specific 'neuro-cardiac junction', where sympathetic neurotransmission occurs in a 'quasi-synaptic' way. The properties of such junctional-type communication fit well with those of the physiological responses elicited by the cardiac sympathetic nervous system, and explain its ability to tune heart function with precision, specificity and elevated temporal resolution., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Optogenetics is a technique exploded in the last 10 years, which revolutionized several areas of biological research. The brightest side of this technology is the use of light to modulate non-invasively, with high spatial resolution and millisecond time scale, excitable cells genetically modified to express light-sensitive microbial ion channels (opsins). Neuroscience has first benefited from such fascinating strategy, in intact organisms. By shining light to specific neuronal subpopulations, optogenetics allowed unearth the mechanisms involved in cell-to-cell communication within the context of intact organs, such as the brain, formed by complex neuronal circuits. More recently, scientists looked at optogenetics as a tool to answer some of the questions, remained in the dark, of cardiovascular physiology. In this review, we focus on the application of optogenetics in the study of the heart, a complex multicellular organ, homing different populations of excitable cells, spatially and functionally interconnected. Moving from the first proof-of-principle works, published in 2010, to the present time, we discuss the in vitro and in vivo applications of optogenetics for the study of electrophysiology of the different cardiac cell types, and for the dissection of cellular mechanisms underlying arrhythmias. We also present how molecular biology and technology foster the evolution of cardiac optogenetics, with the aim to further our understanding of fundamental questions in cardiac physiology and pathology. Finally, we confer about the therapeutic potential of such biotechnological strategy for the treatment of heart rhythm disturbances (e.g. cardiac pacing, cardioversion)., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Morphological and histochemical analysis of the heart is fundamental for the understanding of cardiac physiology and pathology. The accurate detection of different myocardial cell populations, as well as the high-resolution imaging of protein expression and distribution, within the diverse intracellular compartments, is essential for basic research on disease mechanisms and for the translatability of the results to human pathophysiology. While enormous progress has been made on the imaging hardware and methods and on biotechnological tools [e.g., use of green fluorescent protein (GFP), viral-mediated gene transduction] to investigate heart cell structure and function, most of the protocols to prepare heart tissue samples for analysis have remained almost identical for decades. We here provide a detailed description of a novel protocol of heart processing, tailored to the simultaneous detection of tissue morphology, immunofluorescence markers and native emission of fluorescent proteins (i.e., GFP). We compared a variety of procedures of fixation, antigen unmasking and tissue permeabilization, to identify the best combination for preservation of myocardial morphology and native GFP fluorescence, while simultaneously allowing detection of antibody staining toward sarcomeric, membrane, cytosolic and nuclear markers. Furthermore, with minimal variations, we implemented such protocol for the study of human heart samples, including those already fixed and stored with conventional procedures, in tissue archives or bio-banks. In conclusion, a procedure is here presented for the laboratory investigation of the heart, in both rodents and humans, which accrues from the same tissue section information that would normally require the time-consuming and tissue-wasting observation of multiple serial sections.

Starting from the late embryonic development, the sympathetic nervous system extensively innervates the heart and modulates its activity during the entire lifespan. The distribution of myocardial sympathetic processes is finely regulated by the secretion of limiting amounts of pro-survival neurotrophic factors by cardiac cells. Norepinephrine release by the neurons rapidly modulates myocardial electrophysiology, and increases the rate and force of cardiomyocyte contractions. Sympathetic processes establish direct interaction with cardiomyocytes, characterized by the presence of neurotransmitter vesicles and reduced cell-cell distance. Whether such contacts have a functional role in both neurotrophin- and catecholamine-dependent communication between the two cell types, is poorly understood. In this review we will address the effects of the sympathetic neuron activity on the myocardium and the hypothesis that the direct neuro-cardiac contact might have a key role both in norepinephrine and neurotrophin mediated signaling. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aims: Increased cardiac sympathetic neuron (SN) activity has been associated with pathologies such as heart failure and hypertrophy, suggesting that cardiac innervation regulates cardiomyocyte trophism. Whether continuous input from the SNs is required for the maintenance of the cardiomyocyte size has not been determined thus far., Methods and Results: To address the role of cardiac innervation in cardiomyocyte size regulation, we monitored the effect of pharmacological sympathetic denervation in mice on cardiac structure, function, and signalling from 24 h to 30 days in the absence of other pathological stimuli. SN ablation caused an immediate reduction in the cardiomyocyte size with minimal consequences on the resting contractile function. Atrophic remodelling was mediated by the ubiquitin-proteasome system through FOXO-dependent early induction of the muscle-specific E3 ubiquitin ligases Atrogin-1/MAFbx and MuRF1, which was followed by activation of the autophagy-lysosome system. MuRF1 was found to be determinant in denervation atrophy as remodelling did not develop in denervated MuRF1 knock-out (KO) hearts. These effects were caused by decreased basal stimulation of cardiomyocyte β2-adrenoceptor (AR), as atrophy was prevented by treatment of denervated mice with the β2-AR agonist clenbuterol. Consistent with these data, we also observed that β2-AR KO mice showed cardiac atrophy at rest., Conclusion: Cardiac SNs are strong regulators of the cardiomyocyte size via β2-AR-dependent repression of proteolysis, demonstrating that the neuro-cardiac axis operates constitutively for the determination of the physiological cardiomyocyte size. These results are of great clinical relevance given the role of β-AR in cardiovascular diseases and their modulation in therapy.

During the classic "fight-or-flight" stress response, sympathetic nervous system activation leads to catecholamine release, which increases heart rate and contractility, resulting in enhanced cardiac output. Catecholamines bind to β-adrenergic receptors, causing cAMP generation and activation of PKA, which phosphorylates multiple targets in cardiac muscle, including the cardiac ryanodine receptor/calcium release channel (RyR2) required for muscle contraction. PKA phosphorylation of RyR2 enhances channel activity by sensitizing the channel to cytosolic calcium (Ca²+). Here, we found that mice harboring RyR2 channels that cannot be PKA phosphorylated (referred to herein as RyR2-S2808A+/+ mice) exhibited blunted heart rate and cardiac contractile responses to catecholamines (isoproterenol). The isoproterenol-induced enhancement of ventricular myocyte Ca²+ transients and fractional shortening (contraction) and the spontaneous beating rate of sinoatrial nodal cells were all blunted in RyR2-S2808A+/+ mice. The blunted cardiac response to catecholamines in RyR2-S2808A+/+ mice resulted in impaired exercise capacity. RyR2-S2808A+/+ mice were protected against chronic catecholaminergic-induced cardiac dysfunction. These studies identify what we believe to be new roles for PKA phosphorylation of RyR2 in both the heart rate and contractile responses to acute catecholaminergic stimulation.

Doxorubicin (DOXO) remains amongst the most commonly used anti-cancer agents for the treatment of solid tumors, lymphomas, and leukemias. However, its clinical use is hampered by cardiotoxicity, characterized by heart failure and arrhythmias, which may require chemotherapy interruption, with devastating consequences on patient survival and quality of life. Although the adverse cardiac effects of DOXO are consolidated, the underlying mechanisms are still incompletely understood. It was previously shown that DOXO leads to proteotoxic cardiomyocyte (CM) death and myocardial fibrosis, both mechanisms leading to mechanical and electrical dysfunction. While several works focused on CMs as the culprits of DOXO-induced arrhythmias and heart failure, recent studies suggest that DOXO may also affect cardiac sympathetic neurons (cSNs), which would thus represent additional cells targeted in DOXO-cardiotoxicity. Confocal immunofluorescence and morphometric analyses revealed alterations in SN innervation density and topology in hearts from DOXO-treated mice, which was consistent with the reduced cardiotropic effect of adrenergic neurons in vivo. Ex vivo analyses suggested that DOXO-induced denervation may be linked to reduced neurotrophic input, which we have shown to rely on nerve growth factor, released from innervated CMs. Notably, similar alterations were observed in explanted hearts from DOXO-treated patients. Our data demonstrate that chemotherapy cardiotoxicity includes alterations in cardiac innervation, unveiling a previously unrecognized effect of DOXO on cardiac autonomic regulation, which is involved in both cardiac physiology and pathology, including heart failure and arrhythmias. [ABSTRACT FROM AUTHOR]

Optical techniques for recording and manipulating cellular electrophysiology have advanced rapidly in just a few decades. These developments allow for the analysis of cardiac cellular dynamics at multiple scales while largely overcoming the drawbacks associated with the use of electrodes. The recent advent of optogenetics opens up new possibilities for regional and tissue-level electrophysiological control and hold promise for future novel clinical applications. This article, which emerged from the international NOTICE workshop in 20181, reviews the state-of-the-art optical techniques used for cardiac electrophysiological research and the underlying biophysics. The design and performance of optical reporters and optogenetic actuators are reviewed along with limitations of current probes. The physics of light interaction with cardiac tissue is detailed and associated challenges with the use of optical sensors and actuators are presented. Case studies include the use of fluorescence recovery after photobleaching and super-resolution microscopy to explore the micro-structure of cardiac cells and a review of two photon and light sheet technologies applied to cardiac tissue. The emergence of cardiac optogenetics is reviewed and the current work exploring the potential clinical use of optogenetics is also described. Approaches which combine optogenetic manipulation and optical voltage measurement are discussed, in terms of platforms that allow real-time manipulation of whole heart electrophysiology in open and closed-loop systems to study optimal ways to terminate spiral arrhythmias. The design and operation of optics-based approaches that allow high-throughput cardiac electrophysiological assays is presented. Finally, emerging techniques of photo-acoustic imaging and stress sensors are described along with strategies for future development and establishment of these techniques in mainstream electrophysiological research. [ABSTRACT FROM AUTHOR]

Multiphoton microscopy has become essential for dynamic imaging in thick living tissues. High-rate, full-field image acquisition in multiphoton microscopy is achievable by parallelization of the excitation and detection pathways. We developed our approach via a diffractive optical element which splits a pulsed laser into 16 beamlets and exploits a descanned detection system consisting of an array of beamlet-associated photomultiplier tubes. The optical performance of the multiphoton multispot system (MCube) has been characterized in cardiac tissue sections and subsequently used for the first time for fluorescence imaging of cardiomyocyte Ca2+ dynamics in viable acute cardiac slices. Multispot multiphoton microscopy (MMM) has never been used before to monitor Ca2+ dynamics in thick, viable tissue samples. Acute heart slices are a powerful close-to-in vivo model of Ca2+ imaging allowing the simultaneous observation of several cells in their own tissue environment, exploiting the multiphoton excitation ability to penetrate scattering tissues. Moreover, we show that the concurrent high spatial and temporal resolutions afforded by the parallel scanning in MMM can be exploited to simultaneously assess subcellular Ca2+ dynamics in different cells in the tissue. We recorded local Ca2+ release events including macrosparks, travelling waves, and rotors. [ABSTRACT FROM AUTHOR]